Pharmaceutical compositions comprising a modulator of adamts-1

ABSTRACT

The present invention is based on the discovery that the metalloproteinase, ADAMTS-1 (A Disintegrin And Metalloproteinase), is associated with obesity, atherosclerosis, insulin resistance syndrome and non-insulin dependent diabetes. The application is directed to methods for screening for specidic modulators of ADAMS-1 activity, and the use of said modulators for treating the above-mentioned diseases.

[0001] The present invention is based on the discovery that themetalloproteinase, ADAMTS-1 (A Disintegrin And Metalloproteinase), isassociated with obesity, atherosclerosis, insulin resistance syndromeand non-insulin dependent diabetes.

[0002] The ADAM (A Disintegrin And Metalloproteinase) family ofmetalloproteinases, containing 30 members to date, have been identifiedin organisms ranging from yeast to humans (Wolfsberg et al., 1998;Blobel, 1997; Tang, 2001). They have conserved domain structures. ADAMshave been implicated in diverse biological processes, such as sheddingof cell surface molecules and adhesion to cells and matrix proteins. Forexample, ADAM 17 (TACE/TNFα-convertase) cleaves and releases themembrane bound form of TNFα; the Drosophila enzyme kuzbanian and itsmammalian homologue (ADAM 10) have been shown to cleave theextracellular domain of the transmembrane receptor Notch. ADAMs 1 and 2(fertilin α and β) have been shown to be essential for sperm-egg fusionduring fertilization. They have also been shown to be potential playersin pathological events such as cancer metastasis and inflammation.

[0003] Within the past 4 years, a new subset of ADAM-related proteins,known as ADAMTS (A Disintegrin-like And Metalloprotease withThrombospondin type 1 motif), have been identified; there are about 10known members to date, half of which have no known function. The ADAMTS'differ from the previously known ADAMs by the lack of the transmembranedomain and the presence of variable numbers of thrombospondin type I(ITSP-1) repeats. The fist member, ADAMTS-1, was cloned from a mousecachexic colon subline and shown to be inducible by IL-1, suggesting arole as an inflammation associated gene (Juno et al., 1997). It was alsofound up-regulated in the kidney and heart by intravenous administrationof lipopolysaccharide (LPS) in mice, again suggesting the gene may beinduced during inflammatory responses (Kuno et al., 1997; 1998). Theprotein is secreted and binds to extracellular matrix and heparinthrough the thrombospondin and spacer domains (Kuno et al., 1999). HumanADAMTS-1 was identified by a separate group searching for proteinscontaining the anti-angiogenic type 1 repeats of thrombopsondin-1, whichthey called METH-1; the TSP-1 repeats were shown to be required for thepotent anti-angiogenic properties observed (Vazquez et al., 1999).METH-1 has also been described in WO 99/37660 and WO 00/71577(Irulea-Arispe et al). Phylogenetically, ADAMTS-1 has the most homologyto ADAMTS-4 and ADAMTS-8.

[0004] ADAMTS-1-deficient mice have been generated; they are viable butexhibit growth retardation, impaired female fertility, and defects inthe kidney (Shindo et al., 2000). The kidney defect is consistent withthe high levels of expression in the embryonic kidney of normal mice;however, the message levels are significantly reduced in the adult (MRCbiotechnology; Vazquez, 1999). Gon-1, a C. elegans ADAMTS family member,mutants have been generated; they displayed severe defects in gonaddevelopment (Blellock and Kimble, 1999). The distinct phenotypesobserved in ADAMTS-deficient mouse and worm suggest that at least somemembers of this family have some specific and nonredundant roles in cellmigration/remodelling during development. This is in contrast to thatobserved for deficiencies by many metalloproteases, which showsurprisingly mild phenotypes. The effects of the ADAMTS-1 ^(−/−)genotype in obesity, IRS, NIDDM or atherosclerosis were notinvestigated.

[0005] EP 874 050 (SmithKline Beecham/Human Genome Science) concerns thehuman analogue to the mouse ADAMTS-1, in the application designatedintegrin ligand ITGL-TSP. The indications mentioned to be related toADAMTS-1 are limited to angiogenic diseases (cancer, cancer metastasis,chronic inflammatory disorders, rheumatoid arthritis, atherosclerosis,macular degeneration, diabetic retinopathy), restenosis, Alzheimer'sdisease and tissue remodelling.

[0006] WO 98/55643 (Kureha Chemical Industry) from the Kuno group covershuman ADAMTS-1 protein, and its use as an agent for decreasing theleukocyte and thrombocyte blood count and increasing the erythrocyteblood count, e.g. for treatment of inflammatory diseases such asrheumatoid arthritis, hepatitis, nephritis, Crohn's disease, asthma andARDS.

[0007] Recently ADAMTS-1 ^(−/−) mice have been generated by genetargeting. These mice demonstrate a renal phenotype resembling the humanureteropelvic junction obstruction. The effects of the ADAMTS-1 ^(−/−)genotype in obesity, IRS, NIDDM or atheroscleorosis were notinvestigated.

[0008] Presently the degree of information known about the role ofADAMTS-1 in disease has been limited and generally speculative.Therefore is a need for a better understanding of the specific functionof ADAMTS-1 in disease. Furthermore, there is a need for a betterunderstanding of the nature of the underlying physiology of theimportant diseases of obesity, atherosclerosis, IRS and NIDDM. None ofthe known art mentions the connection between the specific level ofADAMTS-1 per se expression and obesity, atherosclerosis or IRS, or thepossibility to prevent or treat these conditions by specificallymodulating the expression level or the activity of ADAMTS-1.

[0009] The present invention is based on the discovery that ADAMTS-1 isspecifically associated with obesity, atherosclerosis, insulinresistance syndrome and non-insulin dependent diabetes.

[0010] According to one aspect of the present invention there isprovided use of a compound able to modulate specifically the activity oramount of ADAMTS-1 in preparation of a medicament for the treatment of adisease indelendently selected from obesity, IRS, NIDDM oratherosclerosis. A preferred use is of a compound able to reducespecifically the activity or amount of ADAMTS-1 in preparation of amedicament for the treatment of obesity, IRS, NIDDM or atherosclerosis.In another embodiment, a preferred use is of a compound able to increasespecifically the activity or amount of ADAMTS-1 in preparation of amedicament for the treatment of obesity, IRS, NIDDM or atherosclerosis.Another embodiment of the invention is use of a compound able to reducespecifically the activity of ADAMTS-1 in preparation of a medicament forthe treatment of obesity, IRS, NIDDM or atherosclerosis. Anotherembodiment of the invention is of a compound able to increasespecifically the activity of ADAMTS-1 in preparation of a medicament forthe treatment of obesity, IRS, NIDDM or atherosclerosis.

[0011] The term “a compound able to modulate specifically the activityor amount of ADAMTS-1” means that the principal pharmaceutical activityrelating to obesity, IRS, NIDDM or atherosclerosis of the compound isdependent on its effect on ADAMTS-1. For example, thiazolidinonecompounds such as for example rosiglitazone, fall outside the scope ofthis definition because they have significant pharmaceutical activitythrough PPAR-γ, see Willson et al (2000), J Med Chem, 43, 527-550.

[0012] In particular, compounds able to modulate specifically the amountof ADAMTS-1 refers to compounds that modulate the amount of ADAMTS-1through a direct effect on the ADAMTS-1 gene or its expression; theADAMTS-1 mRNA, its turn-over, processing, degradation or stability; orthe ADAMTS-1 protein, its turnover, processing, degradation, orstability.

[0013] In particular, compounds able to modulate specifically theactivity of ADAMTS-1 refers to compounds that modulate the activity ofADAMTS-1 without significantly modulating the activity of ADAM 17 (TNF-□converting enzyme, TACE), MMP-1 (interstitial collagenase), MMP-14(membrane type 1-matrix metalloproteinase), MMP-19 (rheumatoidassociated arthritis-associated MMP) and PPAR. However, it iscontemplated that compounds having effects on some of the other ADAMTSs,such as for example the aggrecanases ADAMTS-4 or ADAMTS-5 would fallwithin the definition. Without wishing to be bound by theoreticalconsiderations, it may even be beneficial to have an effect on someother proteins e.g. the aggrecanases.

[0014] The activity of a compound at ADAMTS-1 per se may be measuredthrough a direct effect on the ADAMTS-1 enzyme activity as measured bythe enzyme assays exemplified herein.

[0015] According to another aspect of the present invention there isprovided a method of screening for a compound potentially useful fortreatment of obesity, IRS, NIDDM or atherosclerosis which comprisesassay of the compound for its ability to modulate specifically theactivity or amount ADAMTS-1. Preferably the assay is indelendentlyselected from:

[0016] i) measurement of ADAMTS-1 activity using a cell line whichexpresses ADAMTS-1 or using purified ADAMTS-1 protein; and

[0017] ii) measurement of ADAMTS-1 transcription or translation in acell line expressing ADAMTS-1. Preferably the cell line is a mouse3T3-L1 cell. Preferably the protein is human recombinant ADAMTS-1.

[0018] The amino acid sequence of human ADAMTS-1 can e.g. be obtainedfrom the SwissProt database as id ATS1_HUMAN, DNA sequences encodinghuman ADAMTS-1 can be e.g. obtained from the EMBL database as accessionnos. AP170084, AF060152, AP207664, and AP001697. The amino acid sequenceof mouse ADAMTS-1 can e.g. be obtained from the SwissProt database as idATS1_MOUSE, DNA sequences encoding mouse ADAMTS-1 can be e.g obtainedfrom the EMBL database as accesion nos. AB001735 and D67076.

[0019] According to another aspect of the present invention there isprovided a method of of preparing a pharmaceutical composition whichcomprises:

[0020] i) identifying a compound as useful for treatment of obesity,IRS, NIDDM or atherosclerosis according to a method as described herein;and

[0021] ii) mixing the compound or a pharmaceutically acceptable saltthereof with a pharmaceutically acceptable excipient or diluent.

[0022] The compositions of the invention may be in a form suitable fororal use (for example as tablets, lozenges, hard or soft capsules,aqueous or oily suspensions, emulsions, dispersible powders or granules,syrups or elixirs), for topical use (for example as creams, ointments,gels, or aqueous or oily solutions or suspensions), for administrationby inhalation (for example as a finely divided powder or a liquidaerosol), for administration by insufflation (for example as a finelydivided powder) or for parenteral administration (for example as asterile aqueous or oily solution for intravenous, subcutaneous,intramuscular or intramuscular dosing or as a suppository for rectaldosing).

[0023] The compositions of the invention may be obtained by conventionalprocedures using conventional pharmaceutical excipients, well known inthe art. Thus, compositions intended for oral use may contain, forexample, one or more colouring, sweetening, flavouring and/orpreservative agents.

[0024] Suitable pharmaceutically acceptable excipients for a tabletformulation include, for example, inert diluents such as lactose, sodiumcarbonate, calcium phosphate or calcium carbonate, granulating anddisintegrating agents such as corn starch or algenic acid; bindingagents such as starch; lubricating agents such as magnesium stearate,stearic acid or talc; preservative agents such as ethyl or propylp-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tabletformulations may be uncoated or coated either to modify theirdisintegration and the subsequent absorption of the active ingredientwithin the gastrointestinal track, or to improve their stability and/orappearance, in either case, using conventional coating agents andprocedures well known in the art.

[0025] Compositions for oral use may be in the form of hard gelatincapsules in which the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules in which the active ingredient is mixed withwater or an oil such as peanut oil, liquid paraffin, or olive oil.

[0026] Aqueous suspensions generally contain the active ingredient infinely powdered form together with one or more suspending agents, suchas sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin or condensation products of an alkylene oxide with fatty acids(for example polyoxethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives (such as ethyl orpropyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid),colouring agents, flavouring agents, and/or sweetening agents (such assucrose, saccharine or aspartame).

[0027] Oily suspensions may be formulated by suspending the activeingredient in a vegetable oil (such as arachis oil, olive oil, sesameoil or coconut oil) or in a mineral oil (such as liquid paraffin). Theoily suspensions may also contain a thickening agent such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents such as those set outabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

[0028] Dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water generally contain the activeingredient together with a dispersing or wetting agent, suspending agentand one or more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients such as sweetening, flavouring and colouringagents, may also be present.

[0029] The pharmaceutical compositions of the invention may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, such as olive oil or arachis oil, or a mineral oil, such as forexample liquid paraffin or a mixture of any of these. Suitableemulsifying agents may be, for example, naturally-occurring gums such asgum acacia or gum tragacanth, naturally-occurring phosphatides such assoya bean, lecithin, an esters or partial esters derived from fattyacids and hexitol anhydrides (for example sorbitan monooleate) andcondensation products of the said partial esters with ethylene oxidesuch as polyoxyethylene sorbitan monooleate. The emulsions may alsocontain sweetening, flavouring and preservative agents.

[0030] Syrups and elixirs may be formulated with sweetening agents suchas glycerol, propylene glycol, sorbitol, aspartame or sucrose, and mayalso contain a demulcent, preservative, flavouring and/or colouringagent.

[0031] The pharmaceutical compositions may also be in the form of asterile injectable aqueous or oily suspension, which may be formulatedaccording to known procedures using one or more of the appropriatedispersing or wetting agents and suspending agents, which have beenmentioned above. A sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example a solution in 1,3-butanediol.

[0032] Suppository formulations may be prepared by mixing the activeingredient with a suitable non-irritating excipient which is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Suitable excipientsinclude, for example, cocoa butter and polyethylene glycols.

[0033] Topical formulations, such as creams, ointments, gels and aqueousor oily solutions or suspensions, may generally be obtained byformulating an active ingredient with a conventional, topicallyacceptable, vehicle or diluent using conventional procedure well knownin the art.

[0034] Compositions for administration by insufflation may be in theform of a finely divided powder containing particles of average diameterof, for example, 30μ or much less, the powder itself comprising eitheractive ingredient alone or diluted with one or more physiologicallyacceptable carriers such as lactose. The powder for insufflation is thenconveniently retained in a capsule containing, for example, 1 to 50 mgof active ingredient for use with a turbo-inhaler device, such as isused for insufflation of the known agent sodium cromoglycate.

[0035] Compositions for administration by inhalation may be in the formof a conventional pressurised aerosol arranged to dispense the activeingredient either as an aerosol containing finely divided solid orliquid droplets. Conventional aerosol propellants such as volatilefluorinated hydrocarbons or hydrocarbons may be used and the aerosoldevice is conveniently arranged to dispense a metered quantity of activeingredient.

[0036] For further information on Formulation the reader is referred toChapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (CorwinHansch; Chairman of Editorial Board), Pergamon Press 1990.

[0037] The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. For example, a formulation intended for oraladministration to humans will generally contain, for example, from 0.5mg to 2 g of active agent compounded with an appropriate and convenientamount of excipients which may vary from about 5 to about 98 percent byweight of the total composition. Dosage unit forms will generallycontain about 1 mg to about 500 mg of an active ingredient For furtherinformation on Routes of Administration and Dosage Regimes the reader isreferred to Chapter 25.3 in Volume 5 of Comprehensive MedicinalChemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press1990.

[0038] The size of the dose for therapeutic or prophylactic purposes ofa compound will naturally vary according to the nature and severity ofthe conditions, the age and sex of the animal or patient and the routeof administration, according to well known principles of medicine.

[0039] In using a compound for therapeutic or prophylactic purposes itwill generally be administered so that a daily dose in the range, forexample, 0.5 mg to 75 mg per kg body weight is received, given ifrequired in divided doses. In general lower doses will be administeredwhen a parenteral route is employed. Thus, for example, for intravenousadministration, a dose in the range, for example, 0.5 mg to 30 mg per kgbody weight will generally be used. Similarly, for administration byinhalation, a dose in the range, for example, 0.5 mg to 25 mg per kgbody weight will be used. Oral administration is however preferred.

[0040] The invention is further described in the non-limiting Examplesbelow with reference to the following drawings in which:

[0041]FIG. 1. Real time PCR comparing lean mice, ob/ob mice, and ob/obmice treated with rosiglitazone.

[0042] A comparison of ADAMTS-1 expression in mesenterial fat (5 animalsper group) from lean (1a), untreated ob/ob mice (b) & ob/ob mice treatedwith Rosiglitazone for 7 days (c). ADAMTS-1 mRNA levels aresignificantly elevated in obese (ob/ob) (a) compared with lean (−/ob)animals (b). Treatment with Rosiglitazone decreases the expression inobese mice close to levels found in lean animals (c).

[0043]FIG. 2. Real time PCR analysis of epididymal fat of mice treatedwith rosiglitazone.

[0044] A time course study (3 animals per group) treated withRosiglitazone daily. Real time PCR quantitation of ADAMTS-1 expressionin epididymal fat was performed at 0, 1 3,& 7 days. Expression levelsdecrease substantially in epididymal fat after the first Rosiglitazoneadministration to ob/ob mice, and is further reduced over a 7 dayperiod. The downregulation of ADAMTS-1 precedes the effects on plasmaglucose and triglycerides which are not lowered by the firstadministration of Rosiglitazone.

[0045]FIG. 3. Real time PCR analysis of various tissues.

[0046] Analysis of expression levels of ADAMTS-1 comparing messagelevels in lean compared to ob/ob nmice shows the tissue distribution ofmouse ADAMTS-1. Real time PCR quantitation on pooled cDNA from 3 animalswas normalised against internal control (ribosomal protein 36B4).Expression is up-regulated in several tissues in obese animals. Besidesmesenterial fat, the up-regulation is most pronounced in liver, lung andnotably in aorta

[0047]FIG. 4. ADAMTS-1 message levels in various tissues in treatedmice.

[0048] ADAMTS-1 message levels in various tissues in mice treated withrosiglitazone for 7 days shows a comparison of ADAMTS-1 expression invarious tissues (5 animals per group) from lean (a), untreated ob/obmice (b) & ob/ob mice treated with Rosiglitazone for 7 days (c). Thetissues for each group were as follows, in left to right order bonemarrow; liver, quadriceps; white adipose; and brown adipose.

[0049]FIG. 5. ADAMTS-1 levels in various human tissues

[0050] Real time PCR quantitation on pooled cDNA from severalindividuals was normalised against internal control (ribosomal protein36B4) shows the tissue distribution of human ADAMTS-1. ADAMTS-1expression is significantly higher in the heart than in most othertissues, particularly in the aorta.

[0051]FIG. 6. Immunohistochemistry of Type I aortic lesion.

[0052] Immunohistochemistry with antibodies against ADAMTS-1, α-actin,and macrophage in early fatty streak (type I lesion). A. ADAMTS-1labelling is seen in foam-like cells (arrow) and smooth muscle cells. B.Preabsorption control. C. Macrophage-like immunoreactivity (RAM-56).Some staining colocalize with ADAMTS-1 staining (arrows). D. Smoothmuscle labelling (□-actin).

[0053]FIG. 7. Immunohistochemistry.

[0054] Immunohistochemistry with antibodies against human ADAMTS-1,α-actin, and macrophage in an advanced plaque (type III-IV) in the aorta(A) or coronary artery (B-E). A. ADAMTS-1 like immunoreactivity is seenin the matrix-like core at the base of the aortic plaque (arrow). B.ADAMTS-1 like immunoreactivity at the base of the plaque, close to themedia (arrows). C. Preabsorbed antibody control for ADAMTS-1 staining.D. Macrophage (HAM-56) labelling. E. Actin-like immunoreactivity isfound in the smooth muscle cells of the media and basally in the plaque.Arrows indicate staining co-localize with ADAMTS-1.

[0055]FIG. 8. In situ hybridization.

[0056] In situ hybridization for ADAMTS-1 message in the aorta ofApoE/LDL Receptor—deficient mouse. Note staining is observed both inendothelial and smooth muscle cell layers (arrows). 60×.

[0057]FIG. 9. Expression of hADAMTS-1 in ThP-1 cells.

[0058] Real time PCR analysis for ADAMTS-1 message levels in THP-1 cellstreated with PMA at day 0, 2, 4, 8, 8+24 hours, (8+24 hours with mildlyoxidized LDL).

[0059]FIG. 10. Size exclusion chromatography of proteoglycan from ASMCs.

[0060] A. Analysis of total proteoglycan secreted by primary aorticsmooth muscle cells cultured with 35S and 3H. The two proteoglycanpopulation can be separated according to size by size exclusionchromatography. The large proteoglycan population is made up ofprimarily versican. B. When the total proteoglycan population isincubated with ADAMTS-1 prior to separation by size exclusionchromatography, the size of the large proteoglycan peak is diminishedand the size of the smaller proteoglycan population is increased

[0061]FIG. 11. Real time PCR analysis of aortic SMCs.

[0062] Real time PCR analysis for ADAMTS-1 message levels in A)proliferating primary human aortic smooth muscle cells compared to B)resting confluent human aortic smooth muscle cells.

EXAMPLE 1

[0063] Role of ADAMTS-1 in IRS, NIDDM, Obesity and Atherosclerosis

[0064] 1.1 Materials and Methods Primers H-T₁₁-A SEQ ID NO: 1 H-T₁₁-CSEQ ID NO: 2 H-T₁₁-G SEQ ID NO: 3 H-AP-1 SEQ ID NO: 4 H-AP-2 SEQ ID NO:5 H-AP-3 SEQ ID NO: 6 H-AP-4 SEQ ID NO: 7 H-AP-5 SEQ ID NO: 8 H-AP-6 SEQID NO: 9 H-AP-7 SEQ ID NO: 10 H-AP-8 SEQ ID NO: 11 H-AP-9 SEQ ID NO: 12H-AP-10 SEQ ID NO: 13 Rgh SEQ ID NO: 14 Lgh SEQ ID NO: 15

[0065] Animals, cell culture and treatment. Nine weeks old ob/ob micewere treated for seven days with Rosiglitazone at 30 μmol/kg/day. Thedrug was administrated orally using a gavage. Control animals were fedvehicle (0.1% DMSO). To reduce variation in genetic background malesibling pairs were used with one being treated with drug and the otherwith vehicle. The animals had free access to water and normal mice chow.3T3-L1 cells were grown in 175 cm² flasks to confluency. Dexamethasoneat 2 μg/mL and methylisobutyl-xantine at 0.5 μM were then included inthe media. This treatment was continued for two weeks. This will drivethe differentiation of the cells to adipocytes. Thedexamethasone/methylisobutyl-xantine were removed and cells werethereafter treated with Rosiglitazone at 1 μM for 24 hours. Controlcells were also treated with dexamethasone/methylisobutyl-xantine butwith vehicle instead of Rosiglitazone.

[0066] Tissue isolation and RNA extraction. From treated and controlmice liver, mesenterial fat, epididimus fat, brown fat, white fibersfrom quadriceps (quadri/white), red fibers from quadriceps (quadri/red)and heart were isolated. Care was taken to remove contaminating tissues,blood and hair. All tissues were removed and snap-frozen in liquidnitrogen within 2 minutes after the animal was killed. Tissues wereweighed and RNASTAT-60 (AMS Biotechnology) added. Tissues werehomogenized with a Turrax-blender for one minute on ice. Total RNA wasextracted according to suppliers protocol. Briefly, for tissue amountsup to 100 mg, 1 mL of extraction media was added and the tissuehomogenized. The organic and water phase were separated by acentrifugation. The upper, water, phase, was isolated and RNAprecipitated with one volume of isopropanol. RNA pellet was washed with75% ice-cold ethanol. RNA pellet was dried and dissolved in DEPC treatedwater. For RNA extraction of 3T3-L1 cells the incubation media waspoured off and RNASTAT-60 added. RNA was extracted as described above.To remove residual DNA the total RNA preparation was treated with DNAse.50 μg RNA were incubated at 37° C. with 5 U DNAse (RQ1 DNAse, Promega)in 10 mM CaCl₂; 6 mM MgCl₂; 10 mM NaCl and 40 mM Tris-Cl pH 7.9 in afinal volume of 100 μL. After 15 minutes the reaction was stopped byadding 4 μL 0.5 M EDTA. Protein was removed with aphenol/chloroform/isoamylalcohol extraction. RNA was ethanolprecipitated, re-dissolved in DEPC treated water and quantitated by anOD reading at 260 nm. The quality of the RNA was also checked on an 1%agarose gel.

[0067] Differential display. The differential display was performedusing reagents from GeneHunter Corp. (Nashville, Tenn.) (Liang andPardee, 1992). In three parallel reactions total RNA was reversetranscribed using three different anchored primers, H-T11-A, H-T11-G andH-T11-C. Reactions were performed in duplicate. 0.2 μg of total RNA in13.4 μL water were mixed with 1.6 μL 250 μM dNTP; 4 μL 5× RT buffer (125nmM Tris-Cl pH 8.3, 188 mM KCl, 7.5 mM MgCl₂, 25 mM DTT) and 2 μL 2 μManchored primer. Samples were incubated in a thermocycler, 65° C. for 5min., 37° C. for 60 min. and 75° C. for 5 min. After five minutes at 37°C. M V reverse transcriptase was added. PCRs were set up using the cDNAproduced and combinations of the three anchored primers and tendifferent random primers. For each primer combination the followingreaction was set up. 9.2 μL water, 2 μL 10× PCR buffer (100 mM Tris-ClpH 8.4, 500 mM KCl, 15 mM MgCl2 and 0.01% gelatin); 1.6 μL 25 μM dNTP; 2μL 2 μM anchored primer, 2 μL RT reaction mix; 0.25 μL α-[33P]dATP, 2000Ci/mmol and 0.2 μL AmpliTaq (Perkin-Elmer). Samples were incubated in athermocycler, using the following temperature cycle. 1. 94° C. for 30sec., 2. 40° C. for 2 min., 3. 72° C. for 30 sec., 4. goto step 1, 40cycles and 5. 72° C. for 5 min. After the amplification 3.5 μL of thePCR reaction were mixed with 2 μL loading dye (95% formamide, 10 mM EDTApH 8.0, 0.09% Xylene cyanole FF and 0.09% bromphenol blue). Immediatelybefore loading on 6% PAGE with urea the samples were denaturated at 80°C. PCRs with the same primer combination from treated and untreatedtissues/cells were loaded side by side. When the slower migrating xylenedye reached the bottom of the gel the electrophoresis was stopped. Thegel was transferred to a filter paper and dried in a vacuum gel dryer.Radioactivity was detected by placing the dried gel against a HyperfilmMX™ (Amersham). Bands which appear in the duplicate from one tissue butnot the other were isolated by cutting out the band from the dried gelwith a scalpel. To make sure that the correct band was cut out a secondautoradiography film was exposed to the dried gel. Isolated bands wereboiled in 100 μL water for 15 min. The gel and filter paper were spundown and the supernatant transferred to a fresh tube. The isolatedfragment were re-amplified using the same PCR protocol as describedabove with one change. The dNTP concentration in the re-amplificationwas 20 μM. PCR products were analyzed on 1% agarose. If the PCR gave aproduct of expected size the PCR reaction mixture was used for ligationof the PCR product into the pCRTRAP vector (GeneHunter). Five μL waterwas mixed with 2 μL linearized pCRTRAP; 1 μL 10× ligation buffer(GeneHunter); 2.5 μL PCR product and 0.5 μL T4 DNA ligase (100 U). Theligation reaction was incubated at 16° C. overnight. Ten μL of ligationreaction mixture were transformed into 100 μL GH-competent cells(GeneHunter). Bacteria were plated onto LB with tetracycline (20 μg/mL).Colonies which appeared after overnight incubation at 37° C. werecollected and lysed at 95° C. for 10 min. in 50 μL lysis buffer(GeneHunter). The size of the insert was checked using PCR with a vectorspecific primer pair (Rgh, Lgh). 10.2 μL water were mixed with 2 μL 10×PCR buffer, 1.6 μL 250 μM dNTP; 2 μL 2 μM Lgh primer, 2 μL 2 μM Rghprimer, 0.2 μL AmpliTaq (1 U) and 2 μL colony lysate. PCR were performedat 1. 94° C. for 30 sec., 2. 52° C. for 40 sec., 3. 72° C. for 1 min.,4. goto 1, 40 cycles and 5. 72° C. for 5 min. PCR products was analyzedon 1.5% agarose. In general five positive colonies were used toinoculate 5 mL LB media with tetracycline. Cultures were incubated overnight. Cells were spun down and the pellet used for a Wizard (Promega)plasmid miniprep.

[0068] DNA sequencing. Inserts were sequenced using the Rgh or Lghprimer with the Thermocycler kit for dye terminator cycle sequencing(Perkin Elmer).

[0069] 1.2 Results

[0070] Performance of differential display. In the reverse transcriptionstep, three different anchored primers were used in three independentreactions. These primers have a poly T portion which will hybridize tothe poly A tail in the 3′ end of mRNA. The last base is either a C, G oran A. This procedure subdivides all poly A transcripts in three cDNApools. For each of the cDNA pools PCRs were set up using ten differentrandom primers. This procedure subdivides and amplifies the cDNA poolfurther. The primer combinations used in this study typically generateapproximately 150 fragments/primer combination. Using three differentanchored and ten different random primers a total of 4500 fragments havebeen generated for each tissue. In the literature it has been estimatedthat 15000 genes are expressed at any given moment in a cell (6). Usingthis estimation it can be assumed that ⅓ of the expressed genes in eachtissue has been analyzed. This assumes that each gene will generate onlyone fragment, this may not always be the case. Of the analyzed fragmentsapproximately 150 were detected and isolated as differentiallyexpressed. In the individual tissues 7-23 fragments with differentialexpression were detected with a mean around 15. This indicates thatapproximately 0.1% of expressed genes were affected by the drugtreatment. The highest number of differentially expressed fragments werefound in brown adipose tissue followed by liver, epididimus fat,mesenterial fat, quadri/white, quadri/red and heart. 3T3-L1 cells wereaffected to the same extent as liver in mice. This indicates that of thetissues studied here brown fat is the most affected by the drugtreatment while heart is the least. Liver and fat tissues are moreaffected than the muscle tissues.

[0071] Fragments, up-down regulated by treatment, found in severaltissues. Following drug treatment the levels of expression wereup-regulated for ⅔ of the fragments and down-regulated for the remaning⅓. In all tissues studied both up- and down-regulated expression wereobserved. The highest relative up-regulation of expression was detectedin brown fat while the highest relative down-regulation was detected inheart

[0072] Bioinformatics analysis. The sequences obtained from thedifferential display experiment were compared to sequences found in DNAdatabases. EMBL non-EST, EMBL EST and Patseq were searched using theblastn algorithm. Hits with P(N) values lower than 10-10 in the EMBLnon-EST database were used to identify fragments. Hits from othermammals (than mouse) were used for identification only if thedifferential display fragment aligned to the coding part of thatcDNA/gene. If no hits were obtained in the EMBL non-EST database theEMBL EST database was searched. Only hits from mouse with P(N) lowerthan 1×10¹⁰ were recorded. The patent DNA database PatSeq was searchedfor patented sequences. If no significant non-EST or EST were found thedifferential display fragment was designated unknown. Somewhat less thanhalf of the fragments in this study returned known genes when analyzedagainst databases. One quarter was only identified as ESTs and onequarter as unknown. In all tissues and cells studied here wereapproximately the same proportion between known/EST and unknownobserved.

[0073] 2 Identification of ADAMTS-1 Role in IRS, NIDDM, Obeisty andAtherosclerosis.

[0074] ADAMTS-1 was initially identified in an expression profilingexperiment performed in order to more fully understand the mechanisms ofaction of PPARγ agonists and to find new molecular targets useful fortreatment of insulin resistance syndrome (IRS)/non-insulin dependentdiabetes (NIDDM). The thiazolidinedione (TZD) class of compounds used asinsulin-sensitizing drugs for treatment of non-insulin dependentdiabetes are known to act as ligands for the PeroxisomeProliferator-Activated Receptor γ (PPARγ). A differential displayanalysis was performed using pair wise comparisons of various organs andtissues from control and rosiglitazone-treated (TZD X103, BRL49653,ARH036133) ob/ob mice. These mice are leptin-deficient, obese anddevelop a condition resembling NIDDM with age; some of these symptoms,such as dyslipidemia and obesity, are exhibited by patients who arestatistically likely to develop atherosclerosis. The differentialdisplay analysis resulted in the identification of more than 100 primarysequences derived from known genes, ESTs and unknown genes. Theidentified sequences were run through a confirmation process using realtime quantitative PCR in order to sort out the true up or down regulatedgenes. Also, confirmed hits were further validated in time-course andtissue distribution experiments. Confirmed sequences were taken forbioinformatics and literature studies. 12 potential targets (4 knowngenes, 4 ESTs and 4 previously unknown genes) were selected for furtherstudies.

[0075] One of the differentially expressed sequences corresponded to themouse ADAMTS-1 mRNA, which was significantly elevated in obese ob/obmice compared to lean littermates in mesenterial fat (FIG. 3). ADAMTS-1message was down-regulated after 7 days of Rosiglitazone treatment(administered daily, 30 μmol/kg/day) in epididymal fat tissue (FIG. 2).Real time PCR quantitation on tissues from another set of identicallytreated animals showed that the down-regulation occurred also inmesenterial (FIG. 1) and brown fat tissue (FIG. 4). Observe that themeasurements were done on pooled cDNA from 5 animals, hence the lack oferror bars.

[0076] Subsequently it was found that in humans, the level of ADAMTS-1is higher in the heart compared to most other tissues, particularly inthe aorta (FIG. 5). Since proteases play an integral role both inatherogenesis and for plaque stability by remodelling and degrading ECMproteins, ADAMTS-1 became a potentially interesting target foratherosclerosis.

[0077] Conclusions:

[0078] 1. ADAMTS-1 expression is up-regulated in fat tissue and aorta ofobese (ob/ob) mice, and down-regulated in muscle.

[0079] 2. Treatment of ob/ob mice with PPARγ agonists normalizes theexpression in fat tissue (and to some extent in muscle)

[0080] 3. ADAMTS-1 shows a tissue distribution that is relevant from aNIDDM, obesity and atherosclerosis perspective.

[0081] 4. The gene is expressed (and reacts to PPARγ agonists) in anavailable cell system (mouse 3T3-L1 cells).

[0082] 5. ADAMTS-1 homologues are found in suitable model organisms(mouse, C. elegans and Drosophila).

[0083] 6. ADAMTS-1 belongs to a family of proteases/integrin bindingproteins found to be involved in a multitude of processes in severalimportant diseases.

[0084] 7. The protein is exported, and therefore potentially relativelyeasy to express and purify.

[0085] 8. The ADAMTS-1 molecule has several functional domains (pro-,metalloproteinase-, integrin binding- and matrix binding domains) thatare useful for drug targeting.

[0086] Therefore ADAMTS-1 per se has been demonstrated for the firsttime to be a specific drug target of interest for NIDDM/RS,atherosclerosis and obesity treatment. Without wishing to be bound bytheoretical considerations, its roles might be in tissue/matrixremodelling, differentiation or the release/modification of cytokines,growth factors and receptors.

EXAMPLE 2

[0087] Assay Development

[0088] The complete mouse ADAMTS-1 cDNA has been cloned from epididymalfat tissue and inserted in mammalian expression vectors (for bothconstitutive and inducible expression). The protein is expressed both innative form and with an epitope tag (FLAG) in the C-terminus in order tosimplify detection and purification. The human ADAMTS-1 homologue iscloned and expressed analogously. Antibodies against various functionaldomains in the ADAMTS-1 molecule are contemplated.

[0089] Assays:

[0090] ADAMTS-1 protease activity (cell based or using purifiedrecombinant protein)

[0091] ADAMTS-1 activation (cell based or using purified recombinantprotein) WHAT

[0092] Activation of ADAMTS-1 transcription in suitable cell lines

[0093] Disintegrin assay for monitoring ADAMTS-1l/(target) proteininteractions

[0094] Selection of synthetic substrate through peptide librarytechnology

[0095] Assays for pro-domain cleavage/ADAMTS-1 activation

[0096] Further assay information is presented in Example 9 below.

EXAMPLE 3

[0097] Pharmaceutical Compositions

[0098] The following illustrate representative pharmaceutical dosageforms capable of preparation through the method of the invention asdefined herein (the active ingredient being termed “Compound X”), fortherapeutic or prophylactic use in humans: (a) Tablet I mg/tabletCompound X 100 Lactose Ph.Eur 182.75 Croscarmellose sodium 12.0 Maizestarch paste (5% w/v paste) 2.25 Magnesium stearate 3.0 (b) Tablet IImg/tablet Compound X 50 Lactose Ph.Eur 223.75 Croscarmellose sodium 6.0Maize starch 15.0 Polyvinylpyrrolidone (5% w/v paste) 2.25 Magnesiumstearate 3.0 (c) Tablet III mg/tablet Compound X 1.0 Lactose Ph.Eur93.25 Croscarmellose sodium 4.0 Maize starch paste (5% w/v paste) 0.75Magnesium stearate 1.0 (d) Capsule mg/capsule Compound X 10 LactosePh.Eur 488.5 Magnesium 1.5 (e) Injection I (50 mg/ml) Compound X  5.0%w/v 1 M Sodium hydroxide solution 15.0% v/v 0.1 M Hydrochloric acid (toadjust pH to 7.6) Polyethylene glycol 400  4.5% w/v Water for injectionto 100% (f) Injection II (10 mg/ml) Compound X  1.0% w/v Sodiumphosphate BP  3.6% w/v 0.1 M Sodium hydroxide solution 15.0% v/v Waterfor injection to 100% (g) Injection III (1 mg/ml, buffered to pH6)Compound X  0.1% w/v Sodium phosphate BP 2.26% w/v Citric acid 0.38% w/vPolyethylene glycol 400  3.5% w/v Water for injection to 100% (h)Aerosol I mg/ml Compound X 10.0 Sorbitan trioleate 13.5Trichlorofluoromethane 910.0 Dichlorodifluoromethane 490.0 (i) AerosolII mg/ml Compound X 0.2 Sorbitan trioleate 0.27 Trichlorofluoromethane70.0 Dichlorodifluoromethane 280.0 Dichlorotetrafluoroethane 1094.0 (j)Aerosol III mg/ml Compound X 2.5 Sorbitan trioleate 3.38Trichlorofluoromethane 67.5 Dichlorodifluoromethane 1086.0Dichlorotetrafluoroethane 191.6 (k) Aerosol IV mg/ml Compound X 2.5 Soyalecithin 2.7 Trichlorofluoromethane 67.5 Dichlorodifluoromethane 1086.0Dichlorotetrafluoroethane 191.6 (l) Ointment ml Compound X  40 mgEthanol 300 μl Water 300 μl 1-Dodecylazacycloheptan-2-one  50 μlPropylene glycol to 1 ml

[0099] Note

[0100] The above formulations may be obtained by conventional procedureswell known in the pharmaceutical art. The tablets (a)-(c) may be entericcoated by conventional means, for example to provide a coating ofcellulose acetate phthalate. The aerosol formulations (h)-(k) may beused in conjunction with standard, metered dose aerosol dispensers, andthe suspending agents sorbitan trioleate and soya lecithin may bereplaced by an alternative suspending agent such as sorbitan monooleate,sorbitan sesquioleate, polysorbate 80, polyglycerol oleate or oleicacid.

EXAMPLE 4

[0101] Real Time PCR, Primers and Probes Mouse ADAMTS-1, set 1 Forwardprimer SEQ ID NO: 16 Reverse primer SEQ ID NO: 17 Probe SEQ ID NO: 18Mouse ADAMTS-1, set 2 Forward primer SEQ ID NO: 19 Reverse primer SEQ IDNO: 20 Probe SEQ ID NO: 21 Human ADAMTS-1 Forward primer SEQ ID NO: 22Reverse primer SEQ ID NO: 23 Probe SEQ ID NO: 24

EXAMPLE 5

[0102] Immunohistochemistry

[0103] The following paraffin embedded human material were used. Fattystreak (type I) from a young male and intermediate/advanced aorticplaques (type M-M) were used (courtesy pathology department, SahlgrenskaHospital). Coronary artery was from a female between the ages 40-85. Thesections were stained with eosin and hematoxylin (Cook 1974) to get anoverview of the structure and degree of atherosclerosis.

[0104] To study the presence of smooth muscle cells, a commercial mousemonoclonal antibody against α-actin was used at 1:50 (Cedarlane labs).Another commercial mouse monoclonal antibody against HAM-56 (Daco), wasused at 1:50-1:100 dilution to identify macrophages and possibly foamcells. Two rabbit antibodies raised against the same sequence from humanADAMTS-1 spacer domain were evaluated. Both gave similar results.Preabsorption with ADAMTS-1 peptide was used as control.

[0105] The immunohistochemistry was performed in an immunostainer,Techmate, from Daco. The primary antibodies were incubated on thesections for 12 hours, 25 minutes, followed by washing steps in TRISbuffered saline (RBS). The secondary antibodies weredonkey-anti-rabbit-biotin (Jackson labs) diluted 1:2500 for ADAMTS-1 anddonkey-anti-mouse-biotin (Jackson Labs) diluted 1:1000 for HAM-56 andα-actin. The secondary antibodies were incubated on the sections for 1hours, followed by washing steps. Blockage of the endogenous peroxidaseactivity was performed 3×2.5 minutes with a kit from Daco forBP-blockage. After additional washing steps, HRP was incubated on thesections for 30 minutes, washed and finally the antigen-antibody complexwas visualized by an EAC chromogen kit supplied by Daco for 3×7 minutes.The sections were washed, counterstained in hematoxylin, washed andmounted in Kaisers gelatin glycerine. All sections were examined in aZeiss or an Olympus light microscope.

EXAMPLE 6

[0106] In Situ Hybridization

[0107] Paraffin imbedded aorta from ApoE/LDL Receptor-deficient micewere used for the present study. No lesion was present in the tissueexamined. A 35-S radiolabelled 500 base pair riboprobe was generatedagainst the mouse ADAMTS-1 and used for the present study.

EXAMPLE 7

[0108] Digestion of Proteoglycans

[0109] Human aortic smooth muscle cells were purchased from Clonetics(BioWhittaker) and cultured according to supplier. For preparation oftotal proteoglycan population (total PG), AoSMCs were seeded at 3000cells/cm2 in SmBM2 media (4×80 cm2 flasks). 5 days later, the cells werewashed with Dulbecco's PBS and BME-Diploid medium with FBS was added tothe cells. 1 day later, fresh DME-Diploid medium without FBS, containing35S-Sulfate 33 μCi/mL and 3H-Leucine 17 μCi/mL, 15 mL/bottle andincubated for 3 days. The medium was transferred and dialyzed againstbinding buffer containing 8M urea, 2 mM EDTA, 0.5% Triton X-100, and 20mM Tris-HCl, pH 7.5, for 48 hours and applied to a pre-equilibratedHi-Trap Q column. After washing with 25 mL of Elution buffer A (bindingbuffer+0.25 M NaCl, proteoglycan population is eluted with a linear saltgradient: 0.25-3 M NaCl in binding buffer. Total counts in each fractionwere counted by liquid scintillation counting. The fractions containingPGs were pooled, dialyzed against water, lyophilized and stored at −20degrees C. until use (‘total PG’).

[0110] Separation by size exclusion chromatography was performed using aSuperdex 200 HR 10/30 from Amersham Pharmacia biotech. Total PG samplewas dissolved in 50 mM Tris pH 7.5, 4 M Guanidinium-HCL and loaded on apre-equilibrated column with flow at 0.5 mL/min. 1 mL fractions werecollected and 35S measured for each fraction.

EXAMPLE 8

[0111] Expression of hADAMTS-1 in THP-1 Cells

[0112] THP-1 cells were cultured in RPMI 1640 media supplemented with10% FBS, penicillin-Streptomycin, sodium pyruvate, and nonessentialamino acids (Sigma). PMA (Sigma) was dissolved in ethanol and diluted inthe medium at a final concentration of 160 nM. LDL was equilibrated inPBS and diluted to 400 μg/mL and oxidized with 10 μM CuSO4 for 2.5 hoursat 37 degrees C. The mildly oxidized LDL was equilibrated in RPMI 1640and filter sterilized.

[0113] THP-1 cells were cultured in media containing PMA for 0, 2, 4, 8,and 8+1 days. Another set of plates were incubated with PMA for 8+1days, with the mildly oxidized LDL also present in the last day. RNA wasextracted and ADAMTS-1 message was analyzed by real time PCR.

EXAMPLE 9

[0114] Assay Development

[0115] The complete mouse ADAMTS-1 cDNA has been cloned from epididymalfat tissue and inserted in mammalian expression vectors (for bothconstitutive and inducible expression). The protein is expressed in itsnative form. The human ADAMTS-1 homologue has been cloned and expressedin both its native form and with a cleavable epitope tag (his6) in theC-terminus in order to simplify detection and purification. Antibodiesagainst various functional domains in both the mouse and human ADAMTS-1molecule have been generated.

[0116] Examples of Suitable Assays:

[0117] ADAMTS-1 protease activity (cell based or using purifiedrecombinant protein)

[0118] ADAMTS-1 activation (cell based or using purified recombinantprotein)

[0119] Activation of ADAMTS-1 transcription in suitable cell lines

[0120] Disintegrin assay for monitoring ADAMTS-1/(target) proteininteractions

[0121] Selection of peptide substrate for high-throughput screening.Full length or recombinant metalloprotease domain can be used to screencompounds. Currently, we have a 38 amino acid peptide, derived from thepublished cleavage site on the proteoglycan aggrecan, which can be usedas a substrate suitable for high-throughput screening. Cleavage of thepeptide by recombinant ADAMTS-1 has been confirmed by HPLC analysis. Thepeptide can be labelled with a fluorescence marker to screen by aFRET/quench-type assay. Alternatively, the peptide can be labelled atone end and immobilized to plates or beads at the other end, andcleavage can be monitored by release of labelled cleavage product.Peptide sequence: TSELVEGVTEPTVSQE{circumflex over( )}LGQRPPVTYTPQLFESSGEASC, SEQ ID NO 25: ({circumflex over ( )} denotescleavage site by ADAMTS-1)

[0122] Assays for pro-domain cleavage/ADAMTS-1 activation

[0123] An cell migration assay to measure the activity of ADAMTS-1, suchas migration of aortic smooth muscle cells across a matrix-coatedfilter, is being established. The effect of exogenous recombinantADAMTS-1 will be tested to determine whether higher levels of theprotease can affect migration. When compounds become available, theywill be tested to determine if reduced protease activity can affectactivity.

EXAMPLE 10

[0124] ADAMTS-1: Role of the Protease in Atherosclerosis

[0125] In the human aorta, ADAMTS-1 is expressed normally at levelsbarely detectable by immunohistochemistry in the media but atsubstantially higher levels in foam-like and smooth muscle cells ofearly fatty streaks and in the matrix-like core at the base of typeIII-IV lesions (FIGS. 6,7).). Staining with ADAMTS-1 antibodiesco-localize with smooth muscle cell (□-actin) staining (FIGS. 6a, d and7 a, d). In early fatty streak, ADAMTS-1 staining also co-localizes withstaining observed with the macrophage marker, HAM-56 (FIGS. 6a, c).Preabsorption with peptides used to generate the antibodies removed mostof the staining with the ADAMTS-1 antibodies (FIGS. 6b, 7 b). It hasalso been observed that ADAMTS-1 message is up-regulated substantiallyin human umbilical vein endothelial cells and cardiac microvascularendothelial cells under shear stress, suggesting a potential role inflow-dependent vascular remodelling (Bongrazio et al., 2000). Inaddition, ADAMTS-1 is detected in the aortic plaques of LDLReceptor/ApoE-deficient mice. In situ hybridization experiments suggestthat ADAMTS-1 message is normally expressed by both aortic medial(smooth muscle layer) and endothelial cells of LDLReceptor/ApoE-deficient mice, suggesting that both cell types arecapable of producing ADAMTS-1 FIG. 8). The message levels are low;however, staining was performed on sections of aorta without lesions. Inexperiments with the human monocyte/macrophage-like cell line (THP-1cells), ADAMTS-1 message is induced with PMA, a reagent known to inducedmaturation of monocytes into macrophages, as measured by real time PCR(FIG. 9). Exposure to mildly oxidized LDL did not significantly changethe level of ADAMTS-1 message.

[0126] ADAMTS-1 is able to cleave aggrecan, a proteoglycan containingGAG moieties (chondroitin sulfate); deletion experiments suggest thatbinding to the chondroitin sulfate domain is required for cleavage ofaggrecan (Iozzo, 1998; Kuno et al., 2000; Schwartz et al., 1999). Inaddition, ADAMTS-1 is able to cleave another proteoglycan belonging tothe same gene family, versican, which is expressed at high levelsprimarily by VSMC in atherosclerotic lesions (Evanko et al., 1998; Sandyet al., 2001). See FIG. 10. Total proteoglycan was isolated from primaryaortic smooth muscle cells and incubated with or without ADAMTS-1 priorto separation by size exclusion chromatography. In the presence ofADAMTS-1, the size of the large proteoglycan population made upprimarily of versican is reduced and the size of the peak correspondingto smaller sized proteoglycan population is increased, indicating thatADAMTS-1 was able to cleave and reduce the size of versican.

[0127] The localization of ADAMTS-1 at the base of type III-IV plaques,adjacent to the medial layer, suggests that ADAMTS-1 may play a role inpromoting migration of VSMCs from the vessel wall to the lesions. Realtime PCR (Taqman) analysis for ADAMTS-1 message indicates that it isexpressed at a higher level in proliferating primary aortic VSMCcompared to confluent cells in vitro, consistent with its potential rolein promoting SMC migration in atherosclerosis (FIG. 11). Interestingly,parathyroid hormone-related protein, a hormone expressed by botharterial smooth muscle and endothelial cells and known to be mitogenicwhen targeted to the nucleus, can induced expression of ADAMTS-1 in bone(Miles et al., 2000; Massfelder et al., 1997). A C. elegans member ofthe ADAMTS family, gon-1, has been shown to play an essential role inthe migration of distal tip cells during gonad morphogenesis, presumablyby modifying basement membrane components (Blellock et al., 1999;Blellock and Kimble, 1999). Since a wild-type transgene could rescue themutant phenotype while a protease-defective mutant could not, proteaseactivity is essential for normal distal tip cell migration and gonaddevelopment. Although there is no aggrecan or versican ortholog in C.elegans, there are a number of uncharacterized chondroitin sulfateproteoglycans that may be substrates for GON-1. In addition, bothaggrecan and versican has been shown to have a role in avian neuralcrest migration (Perissinotto et al., 2000). We have generated miceoverexpressing the transgene for ADAMTS-1 as part of plans to furtherinvestigate the role of ADAMTS-1 in atherogenesis.

[0128] In a cross section through a normal aortic vessel, distinctlayers of cells and matrix are observed. Versican binds other ECMproteins, such as tenascin and hyaluronic acid, and cell surfaceglycoproteins (e.g. CDA4-like protein detected on VSMC) and may form ahydrated aggregate which is expandable but resilient, much like aggrecanin cartilage (Hurt-Camejo, 1999). Although VSMC may require thisproteoglycan/ECM-rich environment, the latter may also act as a physicalbarrier that prevents movement. In a normal aorta or vessel, VSMCsprefer to remain in the well delineated medial layer, in the diseasedtissue, however, they migrate into the intima. ADAMTS-1 may be involvedin making the intima more ‘permissive’ for invasion by the VSMCs. Inaddition, cleavage of proteoglycans may lead to the release of growthfactors and cytokines to promote SMC migration from the media to theintima.

REFERENCES

[0129] Blellock, R., Anna-Arriola, SS., Gao, D., Li, Y., Hodgkin, J. andKimble, J. (1999). The gon-1 gene is required for gonadal morphogenesisin Caenorhabditis elegans. Developmental biology 216: 382-393.

[0130] Blellock, R. and Kimble, J. (1999). Control of organ shape by asecreted metalloprotease in the nematode Caenorgabditis elegans. Nature399: 586-590.

[0131] Blobel, C P. (1997). Metalloprotease-Disintegrins: Links to celladhesion and cleavage of TNF α and Notch. Cell 90, 589-592.

[0132] Bongrazio, M., Baumann, C., Zakrzewicz, A., Pries, A R.,Gaehtgens, P. (2000). Evidence for modulation of genes involved invascular adaptation by prolonged exposure of endothelial cells to shearstress. Cardiovascular research 47, 384-393.

[0133] Evanko, S P., Angello, J C., Wight, T N (1998). Formation ofhyaluronan- and versican-rich pericellular matrix is required forproliferation and migration of vascular smooth muscle cells.Arterioscler. Thromb. Vasc. Biol. 19: 1004-1013.

[0134] Hurt-Camejo, E., Rosengren, B., Sartipy, P., Elfsberg, K.,Cajemo, G., Svensson, L. (1999). CD44, a cell surface chondroitinsulfate proteoglycan, mediates binding of interferon-g and some of itsbiological effects on human vascular smooth muscle cells. JBC 274:18597-18964.

[0135] Iozzo, R V. (1998). Matrix proteoglycans: From molecular designto cellular function. Annu. Rev. Biochem. 67: 609-52.

[0136] Kuno, K., Kanada, N., Nakashima, E., Fujiki, F., Ichimura, F.,Matsushima, K (1997). Molecular cloning of a gene encoding a new type ofmetalloproteinase-disintegrin family protein with thrombospondin motifsas an inflammation associated gene. J. Biol. Chem. 272, 556-562.

[0137] Kuno, K. and Matsushima, K (1998). ADAMTS-1 protein anchors atthe extracellular matrix through the thrombospondin type 1 motifs andits spacing region. J. Biol. Chem. 273, 13912-13917.

[0138] Kuno, K., Okada, Y., Kawashima, H., Nakamura, H., Miyasaka, M.,Ohno, H., Matsushima, K. (2000). ADAMTS-1 cleaves a cartilageproteoglycan, Aggrecan. FEBS Letters 478: 241-245.

[0139] Liang, P. and Pardee, A B. (1992) Differential display ofeukaryotic messenger RNA by means of the polymerase chain reaction.Science 257:967-971.

[0140] Massfelder, T., Dann, P., Wu, T L., Vasavada, R., Helwig, J J.,Stewart, AF (1997). Opposing mitogenic and anti-mitogenic actions ofparathyroid hormone-related protein in vascular smooth muscle cells: Acritical role for nuclear targeting. PNAS 94:13630-13635.

[0141] Miles, R R, Sluka, J P, Halladay, D L., Santerre, R F., Hale, LB., Thirunavukkarasu, K, Galvin, R J S., Hock, J M., Onyia, J E. (2000).ADAMTS-1: A cellular disintegrin and metalloprotease with thrombospondinMotifs is a target for parathyroid hormone in bone. Endocrinology141:4533-4542.

[0142] Perissinotto, D., Iacopetti, P., Bellina, L, Doliana, R.,Colombatti, A., Pettway, Z., Bronner-Fraser, M., Shinomura, T., Kimata,K, Mörgelin, M., Löfberg, J., Periis, R. (2000). Avian neural crestmigration is diversely regulated by the two major hyaluronan-bindingproteoglycans PG-M/versican and aggrecan. Development 127: 2823-2842.

[0143] Primakoff, P. and Myles, D G. (2000). The ADAM gene family.Trends in Genetics 16, 83-87.

[0144] Sandy, J D., Westling, J., Kenagy, R D., Iruela-Arispe, U L.,Verscharen, C., Rodriguez-Mazaneque, J C., Zimmermann, D R., Lemire, JM., Fischer, J W., Wight, T N., Clowes, A W. (2001). Versican V1proteolysis in human aorta in vivo occurs at the glu441-ala442 bond, asite that is cleaved by recombinant ADAMTS-1 and ADAMTS4. JBC 276:13372-13378.

[0145] Shindo, T., Kurihara, X, Kuno, K., Yokohama, H., Wada T.,Kurihara, Y., Imai Y., Ogata, M., Nishimatsu, H., Moriyama, N.,Oh-hashi, Y., Morita, H., Ishikawa, T., Nagai, R., Yazaki, Y. andMatsushima, K. (2000) ADAMTS-1: a metalloproteinase-disintegrinessential for normal growth, fertility, and organ morpholoy andfunction. J. Clin. Invest. 105, 1345-1352.

[0146] Schwartz, B N., Pirok, E W., Mensch, J R. and Domowicz, M S.(1999). Domain organization, genomic structure, evolution, andregulation of expression of the aggrecan gene family. Prog. Nuc. Acid.Res. 62: 177-225.

[0147] Tang, B L. and Hong, W. (1999). ADAMTS: A novel family ofproteases with an ADAM protease domain and thrombospondin 1 repeats.FEBS letters 445, 223-225.

[0148] Tang, B L (2001). ADAMTS: A novel family of extracellular matrixproteases. Int. J. Biochem. Cell Biol. 33(1): 3344.

[0149] Wolfsberg, T G, Primakoff, P., Myles, D G., White, J M. (1995).ADAM, a novel family of membrane proteins containing A Disintegrin AndMetalloprotease domain: Multipotential functions in cell-cell andcell-matrix interactions. J. Cell. Biol. 131, 275-278.

[0150] Wolfsberg, T G. and White, J M. (1996). ADAMs in fertilizationand development. Developmental Biology 180, 389-401.

1 15 1 16 DNA Artificial Sequence Description of Artificial SequencePCRprimer 1 aagctttttt ttttta 16 2 16 DNA Artificial Sequence Descriptionof Artificial SequencePCR primer 2 aagctttttt tttttc 16 3 16 DNAArtificial Sequence Description of Artificial SequencePCR primer 3aagctttttt tttttg 16 4 13 DNA Artificial Sequence Description ofArtificial SequencePCR primer 4 aagcttgatt gcc 13 5 13 DNA ArtificialSequence Description of Artificial SequencePCR primer 5 aagcttcgac tgt13 6 13 DNA Artificial Sequence Description of Artificial SequencePCRprimer 6 aagctttggt cag 13 7 13 DNA Artificial Sequence Description ofArtificial SequencePCR primer 7 aagcttctca acg 13 8 13 DNA ArtificialSequence Description of Artificial SequencePCR primer 8 aagcttagta ggc13 9 13 DNA Artificial Sequence Description of Artificial SequencePCRprimer 9 aagcttgcac cat 13 10 13 DNA Artificial Sequence Description ofArtificial SequencePCR primer 10 aagcttaacg agg 13 11 13 DNA ArtificialSequence Description of Artificial SequencePCR primer 11 aagcttttac cgc13 12 13 DNA Artificial Sequence Description of Artificial SequencePCRprimer 12 aagcttcatt ccg 13 13 13 DNA Artificial Sequence Description ofArtificial SequencePCR primer 13 aagcttccac gta 13 14 17 DNA ArtificialSequence Description of Artificial SequencePCR primer 14 gacgcgaacgaagcaac 17 15 17 DNA Artificial Sequence Description of ArtificialSequencePCR primer 15 cgacaacacc gataatc 17

1. UseA method of treating a disease independently selected fromobesity, IRS, NIDDM or atherosclerosis, comprising administering to apatient a therapeutic amount of a compound able to modulate specificallythe activity or amount of ADAMTS-1 in preparation of a medicament forthe treatment of a disease independently selected from obesity, IRS,NIDDM or atherosclerosis.
 2. A usemethod according to claim 1,comprising administering to a patient a therapeutic amount of a compoundable to reduce specifically the activity or amount of ADAMTS-1 inpreparation of a medicament for the treatment of obesity, IRS, NIDDM oratheroscleosis.
 3. A usemethod according to claim 1, comprisingadministering to a patient a therapeutic amount of a compound able toincrease specifically the activity or amount of ADAMTS-1 in preparationof a medicament for the treatment of obesity, IRS, NIDDM oratherosclerosis.
 4. A usemethod according to claim 1, comprisingadministering to a patient a therapeutic amount of a compound able toreduce specifically the activity of ADAMTS-1 in preparation of amedicament for the treatment of obesity, IRS, NIDDM or atherosclerosis.5. A usemethod according to claim 1, comprising administering to apatient a therapeutic amount of a compound able to increase specificallythe activity of ADAMTS-1 in preparation of a medicament for thetreatment of obesity, IRS, NIDDM or atherosclerosis.
 6. A method ofscreening for a compound potentially useful for treatment of obesity,IRS, NIDDM or atherosclerosis which comprises assay of the compound forits ability to modulate specifically the activity or amount of ADAMTS-1.7. A method according to claim 6 in which the assay is independentlyselected from: i) measurement of ADAMTS-1 activity using a cell linewhich expresses ADAMTS-1 or using purified ADAMTS-1 protein; and ii)measurement of ADAMTS-1 transcription or translation in a cell lineexpressing ADAMTS-1.
 8. A method according to claim 7 in which the cellline is a mouse 3T3-L1 cell.
 9. A method according to claim 7 in whichthe protein is human recombinant ADAMTS-1.
 10. A method of preparing apharmaceutical composition which which comprises: i) identifying acompound as useful for treatment of obesity, IRS, NIDDM oratherosclerosis according to a method of any one of claims 6-9; ii)mixing the compound or a pharmaceutically acceptable salt thereof with apharmaceutically acceptable excipient or diluent. cceptable excipient ordiluent.
 11. A method of preparing a pharmaceutical composition whichwhich comprises: i) identifying a compound as useful for treatment ofobesity, IRS, NIDDM or atherosclerosis according to a method of claim 7;ii) mixing the compound or a pharmaceutically acceptable salt thereofwith a pharmaceutically acceptable excipient or diluent.
 12. A method ofpreparing a pharmaceutical composition which which comprises: i)identifying a compound as useful for treatment of obesity, IRS, NIDDM oratherosclerosis according to a method of claim 8; ii) mixing thecompound or a pharmaceutically-acceptable salt thereof with apharmaceutically acceptable excipient or diluent.
 13. A method ofpreparing a pharmaceutical composition which which comprises: i)identifying a compound as useful for treatment of obesity, IRS, NIDDM oratherosclerosis according to a method of claim 9; ii) mixing thecompound or a pharmaceutically acceptable salt thereof with apharmaceutically acceptable excipient or diluent.